Atrial Natriuretic Peptide Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Human Endothelial Cells via Rac1 and NAD(P)H Oxidase/Nox2-Activation

Abstract

Abstract-The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-␣-activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2. T he cardiovascular hormone atrial natriuretic peptide (ANP) plays an important and well-investigated role in the cardiovascular system by participation in blood pressure regulation. 1 ANP mainly acts through binding to the guanylyl cyclase-coupled natriuretic peptide receptor-A (NPR-A), leading to generation of the second messenger cGMP. 1 ANP also binds to NPR-C, which generally acts as a clearance receptor 1 but is also able to mediate an inhibition of adenylyl cyclase activity and activation of phospholipase C. 2 A growing number of studies highlight the profound anti-inflammatory and vasoprotective actions exerted by ANP. 3,4 In this context, we revealed recently that ANP is able to induce mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in cultured endothelial cells (ECs). 5 MKP-1 inhibits tumor necrosis factor-␣ (TNF-␣)-induced p38 MAPK activation, thus leading to a reduction of stress fiber formation, 5 endothelial permeability, 5 and expression of monocyte chemoattractant protein-1. 6 In summary, ANP protects TNF-␣-activated ECs against structural and functional changes by inducing MKP-1.MAPKs play a crucial role in a great variety of intracellular signaling cascades. Pathways of MAPK activation are qu...

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